The focus of this project resides on the role of small antioxidant molecules on the prevention and/or repair of nitrated proteins in mitochondria. Protein nitration occurs in biological systems exposed to nitric oxide. The addition of a nitro group to a tyrosine residue, yielding 3-nitrotyrosine, has been claimed to be a posttranslational modification, part of signal transduction pathways, or a result of "normal" oxidative stress. In this study, we will characterize the mechanism underlying the nitration of proteins in nitric oxide-producing mitochondria, given the important role that these organelles have in the maintenance of cellular ATP, beta-oxidation, heme synthesis, and other functions. Our studies will be aimed at understanding the kinetic and structural changes caused by nitration, and how naturally occurring antioxidants -- present in fruits and vegetables may prevent/reverse this effect. The antioxidant effect of flavonoids, coumarins, and catechins will be studied in terms of hydroxyl substitution, conjugation of rings, and o-methylation of hydroxyl groups to elucidate an association between structure and function. To this end, the specific aims of this proposal are the following: 1. Investigate the molecular mechanisms of mitochondrial protein nitration 2. Elucidate the biological relevance of protein nitration in mitochondria 3. Structure-activity association of naturally occurring flavonoids in the abrogation of protein nitration, and 4. Bioavailability of flavonoids containing diets in nitrative stress.